Cryptococcus neoformans is a leading cause of serious nervous system infections in AIDS patients, but little is known at the biochemical or molecular levels about microbial factors that contribute to its virulence or to the pathogenesis of cryptococcosis. This project focuses on the role of the acyclic polyol mannitol in virulence and pathogenesis. C. neoformans produces large amounts of mannitol in culture and in infected animals. Moreover, a mannitol-hypoproducing C. neoformans mutant that resembles its wild-type parent with respect to several known virulence factors is profoundly hypovirulent in mice. Therefore, the central hypothesis to be examined in this project is that mannitol produced by C. neoformans contributes to virulence and to the pathogenesis of cryptococcosis. This hypothesis will be tested by pursuing two general experimental approaches. In Specific Aim 1, classical genetics will be used to determine if the C. neoformans mutations(s) responsible for mannitol hypoproduction and abnormal growth kinetics at 37 degrees C are linked. Next, extraneous mutations in the mannitol hypoproducing mutant will be removed by repeated backcrossings to a wild-type strain. Lastly, the virulence of the resulting isogenic strains will be compared in mice. In Specific Aim 2, a mannitol-nonproducing C. neoformans mutant will be constructed by disrupting a gene that encodes an enzyme required for mannitol biosynthesis. The gene that encodes mannitol-1-PO4 dehydrogenase (MPD) will be cloned, analyzed and disrupted using an integrative genetic transformation system. Mice and rabbits will be challenged with the resulting null mutant and its wild-type parent to determine if fungal mannitol production contributes directly to virulence and to brain edema formation. Specific Aim 3 is to determine if mannitol produced by C. neoformans protects the fungus from the inhibitory and/or fungicidal effects of human neutrophils, monocytes and macrophages in vitro and of oxidants in cell-free systems. The proposed studies are important because i) they may define a new virulence factor and pathogenetic mechanism in an important infection of AIDS patients ii) they may also apply to diseases caused by other polyol-producing fungal pathogens, iii) they may result in identification and characterization of useful therapeutic targets, iv) they should expand our understanding of C. neoformans at the molecular level, and v) they may provide gene disruption methods that can be used to study other aspects of C. neoformans biology.